Dengue virus, a category A pathogen, infects 40-100 million people each year. Dengue infection severity ranges from the self-limiting Dengue fever to the more lethal forms of the disease, Dengue hemorrhagic fever and Dengue shock syndrome. Although Dengue infections have long been limited to tropical and subtropical areas, the virus has begun to spread outside its former geographic limitations due to the changing ecology of mosquito populations in the face of urbanization and global warming. Furthermore, the boundaries for the four individual serotypes have begun to overlap, putting more people at risk for Dengue hemorrhagic fever, whose incidence is increased upon secondary infection with a different serotype from the primary infection. No treatment or vaccine currently exists. The formation of infectious Dengue virus is critically dependent upon the cellular process of autophagy. Autophagy plays an important role in promoting cell survival under metabolic stress conditions by engulfing cytoplasmic constituents in double-membraned vesicles and mediating their lysosome-dependent degradation. In this capacity, it is not surprising that cellular autophagy can act as a component of the innate immune response, destroying many intracellular pathogens. However, certain microbial pathogens, such as Dengue virus, have been shown to depend on this cellular process, or constituents of the process, for their own propagation. Therefore, inhibitors of autophagy should help to control these infections. The recently published small-molecule inhibitor of autophagy Spautin-1 (specific and potent autophagy inhibitor-1) was found by the laboratory of Junying Yuan (Harvard University) to interfere with the stabilization of beclin-1, required for the formation of autophagy-induction complexes. The Kirkegaard laboratory has shown that Spautin-1 is a potent inhibitor of the assembly of infectious Dengue virions. In the R21 portion of this proposal, the mechanism of this inhibition will be dissected through investigation of the defective viral particles formed in he presence of Spautin-1, genetic analysis of Spautin-resistant viruses, and analysis of primary and secondary infections in mouse models of autophagy perturbation. In the R33 portion, the Yuan and Kirkegaard laboratories will collaborate to test newly developed Spautin-1 derivatives with improved pharmacological properties on Dengue pathogenesis and growth in mice. Although autophagy is a normal constitutive process in all mammalian cells, its temporary inhibition by molecules such as the newly discovered Spautin-1 is likely to allow the clearance of acutely infecting pathogens such as Dengue virus that subvert the cellular autophagy pathway. PUBLIC HEALTH RELEVANCE: Dengue virus infects 40 to 100 million people each year, with outcomes ranging from subclinical fever to hemorrhagic, lethal disease. There is currently no treatment and no vaccine. Furthermore, it is difficult to find drug treatments for RNA viruses such as Dengue virus because they mutate so quickly that drug resistance develops rapidly. However, new compounds that inhibit the process of autophagy (self-eating) in the human cells infected by Dengue inhibit the virus profoundly, because the assembly of viral particles within human cells critically depends on this 'host' function. Work is described to determine exactly how cellular autophagy is used by the virus, and whether drug-resistant viruses will arise frequently if this host process is targeted pharmaceutically.